For more than three decades, research supported by this grant has had a major impact on our understanding of how cells respond to damage to their DNA. It has made particularly important contributions to our knowledge of bacterial SOS responses to DNA damage and the crucial role that translesion DNA polymerases play in DNA damage tolerance and mutagenesis. The proposed research addresses critical problems concerning a newly identified mechanism of stress-induced lethality that results from the incorporation of oxidized deoxynucleotides into DNA, the regulation and function of translesion DNA polymerases, and NusA-dependent mechanisms that couple transcription elongation to DNA repair and damage tolerance. In a completely unanticipated development, our recent research has provided evidence that the incorporation of oxidized deoxynucleotides into DNA contributes significantly to the death of bacterial cells undergoing certain types of stress, which include DinB overproduction, bactericidal antibiotics, and expression of a MalE'-'LacZ fusion protein. This poorly understood type of lethality appears to be directly relevant to cancer and various human diseases. We will carry out in vivo and in vitro experiments to test our model that death results from double stand breaks caused by incomplete base excision repair of nearby incorporated oxidized nucleotides. These should provide important insights to its mechanism, significance, and generality. We have discovered that Y Family DinB-related to DNA polymerases have a striking ability to preferentially carry out accurate translesion synthesis (TLS) over certain adducts typified by N2-furfuryl-dG. We have termed these stealth lesions since the high ratio of DinB to replicative polymerase makes them largely invisible to DNA replication, but they remain in the DNA to cause problems with transcription. Our proposed experiments will yield new mechanistic information about how the dynamic switching between replicative and TLS DNA polymerases at such a lesion is regulated. They will also extend our understanding of the nature of such stealth lesions and of how this capability for specialized bypass relates to DinB's other in vitro and in vivo roles. We have discovered unanticipated roles in DNA repair and damage tolerance for NusA, a component of elongating RNAP polymerases. Our observations have led us to propose a previously unrecognized pathway of NusA-dependent transcription-coupled repair (TCR) that is of particular importance for the removal of stealth lesions from the transcribed strand of expressed genes. Other evidence has led us to suggest a model for NusA-dependent transcription-coupled TLS (TC-TLS) that can help cells deal with transcriptional problems created by gaps in the transcribed strand that result from lesions in the non-transcribed strand. Our proposed experiments will offer new insights to how NusA helps cells repair or tolerate DNA damage.